Introduction

Optimization of cellular transfection and transduction includes choosing a protocol, determining the appropriate mass of plasmid/virus, and evaluating the optimum time after transfection/transduction for the best expression of the construct of interest. Using fluorescent reporters and non-destructive in situ imaging on Celigo cell imaging cytometer, these parameters can be rapidly optimized by repeated imaging of one set of wells or flasks over a period of time. Accurate whole-well bright field cell counting generates cell-based data normalized to absolute cell numbers.

Quantifies fluorescent protein signals on a cell-by-cell basis repeatedly on the same plate providing temporal data

Add propidium iodide for viability of GFP transfect cells in the same well

Imaged and counted cells

(A) Cell image overlay: bright field and green, (B) Celigo image processing software identifies all the cells using bright field image, indicated by the red outline. Within this red outline, green fluorescent intensity was measured to gate cell populations and produce transfection efficiency.

Monitoring transfection rates and viability over 5 days

HeLa cells were transfected with a range of concentrations of a plasmid-encoding turbo-GFP1 and seeded out into a 96-well plate. To monitor cell death, propidium iodide was added to the wells in some experiments. The same plate was imaged daily over a 5-day period.

A single 96-well plate was used to optimize transient transfection of adherent HeLa cells with multiple time points.

Live Whole-Well Transduced Fibroblasts

Live Images: Segmentation of Transduced Fibroblasts

Live images of segmentation using the Expression Analysis application. Nuclei are segmented as the mask (left) and GFP-positive nuclei are segmented as the target (right) to determine transduction efficiency.

Transduction Optimization on Celigo Cell Imaging Cytometer

Images and Analysis of Transduced Human Foreskin Fibroblasts

Images and image analysis of human foreskin fibroblasts transduced with varying amounts of a GFP lentivirus acquired and analyzed on the Celigo cytometer.